dl309 is an adenovirus type 5 (Ad5) mutant that has been extensively utilized for construction of Ad5 mutants in early region 1 (E1), in developing vectors for use as viral vaccines, and in development of gene transfer vectors for gene therapy. Ad5 dl309 has been useful for vector construction because of its altered XbaI restriction pattern and lends itself to a variety of strategies for rescuing inserts or mutations into E1. It contains only one XbaI site at 3.7 map units (m.u.) as compared to wt Ad5 which contains 4(3.7, 29.5, 79.5, and 84.8 m.u.). The loss of the sites at 29.5 and 79.5 m.u. is due to deletions of a few bp but the loss of the site at 84.8 m.u. was the result of a deletion from approximately 83 to 85 m.u. and substitution with a fragment of foreign DNA. Because of the widespread use of dl309 and derivatives of this mutant in the construction of Ad5-based vectors and the need to have precise genetic information on the sequences present in vectors to be used as vaccines and in gene therapy, we have sequenced the alterations in dl309 which affect the XbaI sites at 79.5 and 84.8 m.u. and have determined which E3 proteins are expressed by this virus. The deletion that removes the XbaI site at 84.8 m.u. extends from Ad5 bp 30005-30750 and is substituted with 642-bp of heterologous DNA that shows homology to salmon DNA. This alteration deletes all or part of the coding sequences for the E3 14.7K, 14.5K and 10.4K proteins and these proteins are not detected in dl309 infected cells. The loss of the XbaI site at 79.5 m.u. is the result of a 6-bp deletion which removes two internal amino acids (18 and 19) from the E3 6.7K protein. The E3 6.7K protein and other E3 proteins whose coding sequences are unaffected by the alterations in dl309 (gp19K, 12.5K and 11.6K) were expressed in dl309 infected cells.